Agent for the prevention and treatment of sexually transmitted diseases-I

ABSTRACT

Use of a polylysine, polyamidoamine or polypropylenimine dendrimer having naphthyl disulphonate terminal groups as a topically applied agent in prophylaxis or treatment of sexually transmitted diseases.

FIELD OF THE INVENTION

This invention relates to the prevention and treatment of sexuallytransmitted diseases, and in particular it relates to the use of adendrimer having naphthyl disulphonate terminal groups as a topicallyapplied agent in prophylaxis or therapeutic treatment of these diseases.

BACKGROUND OF THE INVENTION

The global incidence, morbidity, and mortality of sexually transmitteddiseases (STDs) caused by HIV, HSV, and other viral and microbialpathogens is estimated at several hundred millions individualsworldwide. One approach for the control the transmission of STDs is theuse of topically applied, female/male-controlled vaginal, or rectalmicrobicides that inactivate the relevant pathogens. Consequently, thedevelopment of new, safe, topical microbicides for intravaginal orintrarectal use for the prevention and treatment of STDs is an importanttarget for novel drug development.

International Patent Application Nos. PCT/AU95/00350 (WO 95/34595) andPCT/AU99/00763 (WO 00/15240), the contents of which are incorporatedherein by reference, disclose a new class of polyvalent agents—thedendrimers, highly branched macromolecules with a definite envelope ofpolyanionic or cationic surface groups, which have been shown to exhibita range of antiviral and antimicrobial activity with minimal toxicity.Unlike small molecular structures of most antivirals, these dendrimersare a class of polyvalent, highly branched macromolecular compoundsformed by iterative reaction sequences starting from an initial coremolecule with successive layers or stages being added in successive“generations” to build up a three-dimensional, highly ordered polymericcompound. Dendrimers are characterised by the following features: i. aninitiator core which may have one or more reactive sites and bepoint-like or of significant size so as to effect the final topology ofthe dendrimer; ii. layers of branched repeating units attached to theinitiator core; iii. functional terminal groups (such as anionic- orcationic-containing moieties) attached to the surface of the dendrimer,optionally through linking groups.

These macromolecular compounds are synthesised from monomeric buildingblocks with multiple branches or tree-like structures, and the outsidesurface of the molecule carries a number of functional groups that leadto recognition by a biological receptor.

The preparation of dendrimers is well known, and is described by way ofexample in U.S. Pat. Nos. 4,289,872 and 4,410,688 (describing dendrimersbased on layers of lysine units), as well as U.S. Pat. Nos. 4,507,466,4,558,120, 4,568,737 and 4,587,329 (describing dendrimers based on otherunits including polyamidoamine or PAMAM dendrimers).

In antiviral and antimicrobial testing, a subset of these dendrimerstructures have unexpectedly shown exceptional activity against a broadspectrum of microorganisms associated with sexually transmitteddiseases, that makes them agents of choice for the development of avaginal or rectal microbicide for the prophylaxis and treatment ofsexually transmitted diseases.

SUMMARY OF THE INVENTION

According to the present invention, there is provided a compound of theformula I, II, or III:

wherein R represents a group of the formula IV:

or a pharmaceutically acceptable salt thereof.

Suitable pharmaceutically acceptable base addition salts include, butare not limited to, metallic salts such as the aluminium, calcium,lithium, magnesium, potassium, sodium and zinc salts, as well as organicsalts made from organic amines such as N,N′-dibenzyl-ethylenediamine,chloroprocaine, diethanolamine, ethylenediamine, dicyclohexylamine,meglumine (N-methylglucamine) and procaine, quaternary amines such ascholine, and sulphonium and phosphonium salts.

Particularly preferred compounds of the present invention are thecompounds referred to herein as SPL-7013, SPL-7304 and SPL-7320 thestructures of which consist of polylysine dendrimer, polyamidoamine(PAMAM) dendrimer, and polypropylenimine dendrimer scaffoldsrespectively with the active surface groups consisting of 32 naphthyldisulphonic acid groups as sodium salts. Each of thenaphthyl-disulphonate functional surface groups is attached to thebranched dendrimer scaffold with an amido-methyleneoxy linkage to the 32terminal groups.

The present invention also provides a topical pharmaceutical compositionfor prophylactic or therapeutic treatment of sexually transmitteddiseases in a human patient, which comprises a compound of the formulaI, II, or III above or a pharmaceutically acceptable salt thereof, inassociation with at least one pharmaceutically acceptable, topicalcarrier or diluent.

In another aspect, the present invention also provides a method for theprophylactic or therapeutic treatment of sexually transmitted diseasesin a human patient, which comprises topical administration to thepatient of an effective amount of a compound of the formula I, II, orIII above or a pharmaceutically acceptable salt thereof.

In yet another aspect, the invention provides use of a compound of theformula I, II, or III above or a pharmaceutically acceptable saltthereof in the manufacture of a medicament for topical administration inthe prophylactic or therapeutic treatment of sexually transmitteddiseases in a human patient.

DETAILED DESCRIPTION OF THE INVENTION

Compounds of the formula I, II, and III, and their pharmaceuticallyacceptable salts are novel compounds which have unexpectedly shownexceptional activity against a broad spectrum of pathogens associatedwith sexually transmitted diseases.

As described above, the compounds SPL-7013, SPL-7304, and SPL-7320 arepreferred compounds of the present invention, and have been found toexhibit significant antiviral activity, particularly against viral andmicrobial vectors of the most common sexually transmitted diseases.

SPL-7013 exhibits a broad-spectrum antiviral activity with high efficacyand minimal cell or animal toxicity against vectors of several of themost important vaginally or rectally sexually transmitted diseases. Highactivity has been determined against genital Herpes virus-2 (HSV-2) bothin in vitro cell tests and in vivo in an animal (mouse) model test andin vitro cell tests against Herpes virus-1 (HSV-1) and HumanImmunodeficiency viruses (HIV-1, and HIV-2). It has also been shown tobe active against the causative agent of genital warts, HumanPapillomavirus (HPV), and against the bacterial vector of non-specificurethritis, Chlamydia trachomatis. In cell tests, SPL-7013 has alsoshown activity against viral strains of Herpes virus-2 resistant tocurrently used modified nucleoside based antiviral agents. In additionSPL-7304 and SPL-7320 show high activity against HSV-1, HSV-2, HIV-1,and HIV-2. Furthermore SPL-7013, SPL-7304, and SPL-7320 is active inCD4-dependant and CD4-independent HIV transmission assays, and iseffective at preventing HIV-1 attachment and fusion. All compounds havebeen shown not to inhibit the growth of various species of beneficialLactobacillus. In addition SPL-7013, SPL-7304, and SPL-7320 have beenshown to be effective in the prevention of infection of human peripheralblood monocular cells (PBMCs) with either HIV-1 RoJo or SIV 89.6 pd.

Accordingly, these compounds are useful in prophylaxis and therapeutictreatment of sexually transmitted diseases as topical microbicide agentsintended for application to the vaginal or rectal mucosa to protectagainst sexually transmitted infections.

The present invention also provides a topical pharmaceutical compositionfor prophylactic or therapeutic treatment of sexually transmitteddisease in a human patient, which comprises a compound of formula I, II,or III or salt thereof as described above, in association with at leastone pharmaceutically acceptable, topical carrier or diluent.

The formulation of such compositions is well known to persons skilled inthis field. Suitable pharmaceutically acceptable carriers and/ordiluents include any and all conventional solvents, dispersion media,fillers, solid carriers, aqueous solutions, coatings, antibacterial andanti fungal agents, isotonic, and absorption enhancing or delayingagents, activity enhancing or delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art, and it is described, by way of example, in Remington'sPharmaceutical Sciences, 18th Edition, Mack Publishing Company,Pennsylvania, USA. Except insofar as any conventional carrier and/ordiluent is incompatible with the active ingredient, use thereof in thepharmaceutical compositions of the present invention is contemplated.Supplementary active ingredients including agents having antiviral orantimicrobial activity can also be incorporated into the compositions ofthis invention.

It is especially advantageous to formulate compositions in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform as used herein refers to physically discrete units suited asunitary dosages for the human subjects to be treated; each unitcontaining a predetermined quantity of active ingredient calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier and/or diluent. The specifications for the noveldosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active ingredient andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active ingredientfor the particular treatment.

As previously described, the present invention also provides a methodfor the prophylactic or therapeutic treatment of sexually transmitteddiseases in a human patient by topical administration to the patient ofan effective amount of a compounds of formula I, II or III or saltthereof as described above. In addition, the present invention providesthe use of a compound of formula I, II, or III or salt thereof asdescribed above in the manufacture of a medicament for topicaladministration in such prophylactic or therapeutic treatment of sexuallytransmitted diseases.

A variety of topical administration routes are available. The particularmode selected will depend, of course, upon the particular conditionbeing treated and the dosage required for prophylactic or therapeuticefficacy. The methods of this invention, generally speaking, may bepracticed using any mode of administration that is medically acceptable,meaning any mode that produces prophylactic or therapeutic levels of theactive component of the invention without causing clinicallyunacceptable adverse effects. Such modes of administration include thevaginal, rectal, and trans-dermal routes. Suitable formulations fortopical, particularly vaginal or rectal, administration includesolutions, suspensions, gels, lotions and creams as well as discreteunits such as suppositories and microencapsulated suspensions. Otherdelivery systems can include sustained release delivery systems whichcan provide for slow release of the active component of the invention,including sustained release gels, creams, suppositories, or capsules.Many types of sustained release delivery systems are available. Theseinclude, but are not limited to: (a) erosional systems in which theactive component is contained within a matrix, and (b) diffusionalsystems in which the active component permeates at a controlled ratethrough a polymer.

The active component of the present invention is administered inprophylactically or therapeutically effective amounts. Aprophylactically or therapeutically effective amount means that amountnecessary at least partly to attain the desired effect, or to delay theonset of, inhibit the progression of, or halt altogether, the onset orprogression of the particular condition being treated. Such amounts willdepend, of course, on the particular condition being treated, theseverity of the condition and individual patient parameters includingage, physical condition, size, weight and concurrent treatment. Thesefactors are well known to those of ordinary skill in the art and can beaddressed with no more than routine experimentation. It is preferredgenerally that a maximum dose be used, that is, the highest safe doseaccording to sound medical judgment. It will be understood by those ofordinary skill in the art; however, that a lower dose or tolerable dosemay be administered for medical reasons, psychological reasons or forvirtually any other reasons.

Generally, at intervals to be determined by the prophylaxis or treatmentof pathogenic states, intra-vaginal or intra-rectal doses of activecomponent will be from about 0.01 mg/kg per day to 1000 mg/kg per day.Small doses (0.01-1 mg) may be administered initially, followed byincreasing doses up to about 1000 mg/kg per day. In the event that theresponse in a subject is insufficient at such doses, even higher doses(or effective higher doses by a different, more localised deliveryroute) may be employed to the extent patient tolerance permits. Multipledoses per day can be contemplated to achieve appropriate systemic levelsof compounds.

In a particularly preferred aspect, the present invention provides theuse of SPL-7013, SPL7304 or SPL7320 as a broad spectrum vaginal orrectal microbicide useful in the prevention and treatment of viral andmicrobial sexually transmitted diseases. SPL-7013, SPL7304 and SPL7320are water soluble and can be used in solution, or formulated in asuitable vehicle in the form of gel, lotion, cream or suppository ormicroencapsulated suspension in aqueous or non-aqueous solvents,together with enhancers or delayers of its activity, agents for itsenhanced or delayed absorption on topical application, or agents toenhance adhesion to vaginal/rectal epithelial or mucosal layers.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated step or steps or integer or group of integers but not theexclusion of any other step or steps or integer or group of integers.

Further features of the present invention will be apparent from thefollowing Examples which are included by way of illustration, notlimitation, of the invention.

EXAMPLE 1 A. Preparation of BHAlyslys₂lys₄lys₈lys₆ TFA₃₂ 1.N,N′-Di-t-Boc-L-Lysine (DBL)

L-Lysine.HCl (1.83 kg; 10 mol) was dissolved in a solution of sodiumhydroxide (880 g; 22 mol) in water (20 l) and the resulting solutiondiluted with t-butanol (15 l). Di-t-butyl dicarbonate (4.48 kg; 20.5mol) was then added dropwise over 1 hour during which time the reactionmixture became milky and warm (30-35° C.). The mixture was left to stirovernight to give a clear solution. The solution was extracted withpetroleum (40-60° C.) (2×10 l) and the organic phase extracted with sat.NaHCO₃ solution (3×4 l). The combined aqueous layers were cooled in anice bath and acidified carefully to pH 2 with 1M KHSO₄ solution. Theturbid mixture was then extracted with ether (4×16 l) and the combinedorganic layers washed with water (2×8 l), dried (MgSO₄) and concentratedwith a bath temperature of <30° to give a quantitative yield ofN,N′-di-t-Boc-L-Lysine as a very viscous oil.

2. N,N′-Di-t-Boc-L-Lysine 4-nitrophenyl ester (DBLONp)

A solution of dicyclohexylcarbodiimide (DCC) (2.06 kg; 10 mol) in ethylacetate (5 l) was added dropwise to an ice-cold stirred solution ofDi-t-Boc-L-lysine (DBL) (3.46 kg; 10 mol) and 4-nitrophenol (NpOH) (1.39kg; 10 mol) in ethyl acetate (15 l) under nitrogen. After the additionwas complete the mixture was allowed to warm to room temperature andstirred overnight. The resulting white suspension was then filtered andthe filtrate concentrated to give a yellowish solid residue. The residuewas recrystallised from ether to give N,N′-di-t-Boc-L-lysine4-nitrophenyl ester as a white solid (yield ca. 60%). More of theproduct can later be isolated from the mother liquors.

3. BHAlys.2HCl

A solution of DCC (41.2 g; 0.2 mol) in dry dichloromethane (200 ml) wasadded to an ice-cold solution of DBL (69.2 g; 0.2 mol) andbenzhydrylamine (BHA) (36.65 g; 0.2 mol) in dichloromethane (600 ml).The mixture was stirred at 0° for 30 minutes and then at roomtemperature for 3 hours. The resulting suspension was filtered and a tlc(pet.ether/EtOAc, 9:1) of the filtrate showed no starting material. Thefiltrate was washed with 5% HCI, water, sat. NaHCO₃ and water; thendried (MgSO₄) and concentrated to give a foam.

Trifluoroacetic acid (TFA) (400 ml) was added to a stirred solution ofthe foam in dry dichloromethane (200 ml) at room temperature. There wasinitially vigorous gas evolution which stopped after ca 15 minutes; tlc(EtOAc) showed no starting material. The solution was stirred for anadditional 2 hours and then concentrated to give a brownish oil. Thisoil was dissolved in dry acetonitrile (600 ml) and a saturated solutionof HCl gas dissolved in absolute ethanol (ca. 360 ml) added withswirling. A white solid soon began to crystallise and the mixture wasleft to stand for 1 hour at room temperature to completecrystallisation. The mixture was filtered and the solid washed with dryacetonitrile, then dried to give BHAlys.2HCl as a white powder (yieldca. 80%).

4. BHAlyslys₂Boc₄

Triethylamine (39.5 ml; 0.283 mol) was added to a stirred solution ofBHAlys.2HCl (54.4 g; 0.14 mol) in dry DMF (300 ml). A white suspensionformed which was stirred for 15 minutes when DBLONp (262 g; 0.56 mol)was added. The mixture immediately became yellow and was stirred for 3hours, maintaining the pH of the mixture at 8-9 by addition oftriethylamine. The reaction mixture was then added slowly to a largevolume of vigorously stirred water and the mixture stirred overnight.The precipitate was collected by filtration and washed with water (3×)and dried to give a yellowish solid. This solid was powdered and thenwashed successively with ether until the ether showed no yellow colouron treatment with aqueous NaOH. The remaining solid was dried to giveBHAlyslys₂Boc₄ as a white powder (yield ca. 70%).

5. BHAlyslys₂lys₄ Boc₈

Trifluoroacetic acid (600 ml) was added to a solution ofBHAlyslys₂Boc₄(116 g; 0.12 mol) in dry dichloromethane (600 ml) andthere was an immediate vigorous evolution of gas. The solution wasstirred for 2 hours and the concentrated to give a viscous oil. This oilwas dissolved in dry DMF (500 ml) and the pH of the solution adjusted to8-9 with triethylamine. DBLONp (460 g; 0.99 mol) was then added and theyellow solution stirred for 2 days at room temperature with periodic pHadjustment with triethylamine to maintain the pH above 8. The reactionwas then precipitated into water and worked up as described above togive BHAlyslys₂lys₄Boc₈ as a white powder (yield ca. 100%).

6. BHAlyslys₂lys₄lys₈Boc₁₆

Trifluoroacetic acid (1 l) was added to a solution of BHAlyslys₂lys₄Boc₈(200 g; 0.106 mol) in dry dichloromethane (1 l) and there was animmediate vigorous evolution of gas. The solution was stirred for 2hours and the concentrated to give a viscous oil. This oil was dissolvedin dry DMF and the pH of the solution adjusted to 8-9 withtriethylamine. DBLONp (782 g; 1.67 mol) was then added and the yellowsolution stirred for 2 days at room temperature with periodic pHadjustment with triethylamine to maintain the pH above 8. The reactionwas then precipitated into water and worked up as described above togive BHAlyslys₂lys₄lys₈Boc₁₆ as a white powder (yield ca. 100%).

7. BHAlyslys₂lys₄lys₈ lys₁₆ Boc₃₂

Trifluoroacetic acid (1.6 l) was added to a mixture ofBHAlyslys₂lys₄lys₈Boc₁₆ (300 g; 0.081 mol) in dry dichloromethane (800ml) and there was an immediate vigorous evolution of gas. The solutionwas stirred for 2 hours and the concentrated to give a viscous oil. Thisoil was dissolved in dry DMF (1.2 l) and the pH of the solution adjustedto 8-9 with triethylamine. DBLONp (1030 g; 2.21 mol) was then added andthe yellow solution stirred for 2 days at room temperature with periodicpH adjustment with triethylamine to maintain the pH above 8. Thereaction was then precipitated into water and worked up as describedabove to give BHAlyslys₂lys₄lys₈lys₁₆Boc₃₂ as a white powder (yield ca.100%).

8. BHAlyslys₂lys₄lys₈lys₁₆TFA₃₂

Trifluoroacetic acid (168 ml) was added in one portion to a suspensionof the t-butyloxycarbonate (Boc) protected polylysine coreBHAlyslys₂lys₄lys₈lys₁₆Boc₃₂ (20 g) in dichloromethane (350 ml) and thereaction was stirred for 14 hours at ambient temperature. The reactionmixture was concentrated in vacuo, dissolved in H₂O (200 ml) and freezedried to give BHAlyslys₂lys₄lys₈lys₁₆TFA₃₂ (17.5 g, 83%) as an off-whitesolid.

B. Preparation of SPL 7013 (i)Disodium-[1-(oxymethylene-carboxyethyl)-3,6-naphthalene-disulphonate]

Disodium 1-hydroxy-3,6-naphthalene-disulphonate (100 g) was dissolved indimethyl sulfoxide (250 ml) and diisopropylethylamine (60 ml) was addedin one portion. Ethyl bromoacetate (38 ml) was added over a 30 minperiod and the reaction was stirred for 14 hours at ambient temperaturewith the exclusion of moisture. The reaction was slowly poured intoethyl acetate (2 L) to give an oily residue/gum. The supernatant wasdecanted and additional ethyl acetate (1.5 L) was added. Severaltriturations followed by washing with acetone of the residue gavedisodium-[1-(oxymethylene-carboxyethyl)-3,6-naphthalene-disulphonate](97 g, 78%) as a brown solid.

(ii) 1-(oxymethylene-carboxy)-3,6-naphthalene-disulphonic acid

Aqueous NaOH (134 ml) was added in one portion to a solution ofdisodium-[1-(oxymethylene-carboxyethyl)-3,6-naphthalene-disulphonate](97 g) in H₂O 500 ml. The reaction was stirred for 14 hours at ambienttemperature and then passed through an IR120 (acid form) column. Freezedrying the collected fractions gave1-(oxymethylene-carboxy)-3,6-naphthalene-disulphonic acid (67 g, 83%) asa brown solid.

(iii) BHAlyslys₂lys₄lys₈lys₁₆[NH—CO—CH₂O-3,6-Naphthyl-(SO₃Na)₂]₃₂—SPL7013

1-(oxymethylene-carboxy)-3,6-naphthalene-disulphonic acid (47.85 g) wasdissolved in DMF (500 ml) and diisopropylethylamine (115 ml) was addedin one portion. This solution was then added to a solution prepared byadding benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate (PyBOP; 72 g) in one portion to a solution ofBHAlyslys₂lys₄lys₈lys₁₆TFA₃₂ (15.95 g) in DMF (650 ml). DMF (100 ml) wasused to rinse the dropping funnel. The reaction mixture was stirred for14 hours at ambient temperature and then diluted with water (10 L). Thesolution was pumped through a 1 micron depth filter and across a PallFiltron apparatus (Pall Gelman) containing a 10 kD membrane. Theresulting solution was passed through an IR120 (sodium form) column,pumped through a 0.22 micron depth filter and freeze dried to giveSPL-7013 as an off-white solid in a 75% yield. Retention time=15 min onan ODS-EC 15 cm×4.6 mm HPLC column at 30° C. with the detectionwavelength set at 240 nm. The sample was eluted at a flow rate of 1 mlper minute using the gradient elution detailed below: Time mins % A % B0 90 10 2 90 10 20 10 90 30 10 90 31 90 10Where solvent A=0.015M TBAH in 0.01M NH₄OAc, (pH 7.0) and solventB=0.015M TBAH in MeOH.

Mass spectral analysis of SPL7013 indicate a molecular weight of16,580±2 Dalton. The expected average molecular weight expected forSPL7013, molecular formula, C₅₈₃H₅₇₇N₆₃Na64O₂₈₇S₆₄ is 16581.53.

C. Preparation of SPL 7304

In a similar way SPL7304 was prepared by the PyBOP coupling of1-(oxymethylene-carboxy)-3,6-naphthalene-disulphonic acid withpolyamidoamine (PAMAM) dendrimer, generation 3 (Starburst®,Sigma-Aldrich Pty. Ltd., Australia).

D. Preparation of SPL 7320

In a similar way, SPL7320 was prepared by the PyBOP coupling of1-(oxymethylene-carboxy)-3,6-naphthalene-disulphonic acid withpolypropylenimine dotriacontaamine dendrimer, generation 4.0 (DAB-Am-32,Sigma-Aldrich Pty. Ltd., Australia).

EXAMPLE 2 Biological Activity of SPL 7013, SPL 7304, and SPL 7320 A.Activity Against Human Immunodeficiency Virus

The human immunodeficiency virus strains used were HIV-1 (IIIB)¹ andHIV-2 (ROD)². Anti-retroviral activity and cytotoxicity measurementswere carried out in parallel. They were based on the viability of MT-4cells that had been infected with HIV and then exposed to variousconcentrations of the test compounds. After the MT-4 cells were allowedto proliferate for 5 days, the number of viable cells was quantified bya tetrazolium-based colorimetric3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)procedure in 96-well microtrays.³

In all of these assays, viral input (viral multiplicity of infection,MOI) was 0.01, or 100 times the 50% cell culture infective dose(CCID₅₀). The 50% antivirally inhibitory concentration (EC₅₀) wasdefined as the compound concentration required to protect 50% of thevirus-infected cells against viral cytopathicity. The 50% cytotoxicconcentration (CC₅₀) was defined as the compound concentration requiredto reduce the viability of mock-infected cells by 50%. The >symbol isused to indicate the highest concentration at which the compounds weretested and still found non-cytotoxic. Average EC₅₀ and CC₅₀ values forseveral separate experiments are presented as defined above. As a rule,the individual values did not deviate by more than 2-fold up or downfrom the EC₅₀ and CC₅₀ values. SI is the antiviral selectivity indexCC₅₀/EC₅₀. TABLE HIV Activity Compound Strain EC50 μg/ml EC90 μg/ml CC50μg/ml SI SPL-7013 IIIB 0.45 0.954 >125 >280 ROD 3.26 4.86 >50 >17SPL-7304 IIIB 0.15 >88 >652 ROD 0.8 >50 >63 SPL-7320 IIIB 0.164 >88 >537ROD 1.153 >50 >47IIIB = HIV-1 ROD = HIV-2EC₉₀ = Effective concentration to reduce viral plaque 90%.

(i) Methods for the CD4-dependent and CD4-independent HIV TransmissionInhibition Assays, HIV Attachment and Fusion, and Inhibition ofLactobacillus sp. Growth Cells and Viruses

HIV-1_(IIIB), and the, HeLa CD4 LTR β-gal, GHOST X4/R5, and HL2/3 celllines were obtained from the NIH AIDS Research and Reference ReagentProgram (Bethesda, Md.), and maintained as recommended. ME180 cells wereobtained from the American Type Culture Collection (Manassas, Va.).

Test Material Handling and Storage

Compounds are typically solubilized in 100% DMSO and stored at −80° C.until tested, unless alternative solvents and storage conditions arespecified by the sponsor. Frozen stocks are thawed at room temperature,pre-warmed for 15 min at 37° C. and vortexed prior to preparation ofworking solutions in tissue culture medium. During all stages ofcompound dilution and handling, compounds are protected from incidentallight by opaque coverings and by storage and dilution in opaque oramber-colored tissue culture plastics. Additionally, incidental room andlaminar flow tissue culture hoods light exposure is controlled byreducing total fluorescent lighting in the laboratory by 50%. The finalDMSO concentration is 0.25% at the highest test concentration.

CD4-Independent HIV Transmission Inhibition Assay

ME180 cells, a CD4 negative, X4 positive cervical epithelial cell lineis maintained in RPMI 1640 supplemented with 10% fetal bovine serum,glutamine and antibiotics. Twenty-four h prior to the assay ME180 cellsare trypsonized, washed and seeded in 96-well flat bottom microtiterplates at a density of 2×10³ cells per well. On the day of the assay, H9cells chronically infected with the SKI clinical isolate of HIV-1([9-SK1) are treated with freshly made mitomycin C (200 μg/ml) for 60minutes at 37° C. This concentration of mitomycin C is sufficient toresult in cell death, but allows virus transmission to occur. Aftermitomycin C treatment the H9-SK1 cells are washed three times withtissue culture medium. Test compounds are added to the ME180 monolayerfollowed by 2×10⁴ H9-SK1 cells. The ME180 cells are co-cultured withH9-SK-1 cells and test material for 4 h, and the H9-SK1 cells areremoved by washing the ME180 monolayer three times with PBS. At 24 and48 h post assay initiation the wells are again washed three times withPBS to ensure removal of the H9-SK1 cells, and culture continued in testmaterial free media. At six days post-co-cultivation, supernatants arecollected and evaluated for p24 antigen expression by ELISA. Cellviability is assessed by XTT dye reduction. Compounds which are judgedas active in the first test are retested with or without the addition ofmucin. Testing in the presence of mucin is carried out by addition of200 μg/ml of porcine mucin (Sigma Chemical Co., St Louis, Mo.) to thetransmission reactions. Transmission intervals and washing withoutreplacement of mucin or compound are carried as described above. Alldeterminations are performed in triplicate with serial ½ Log₁₀ dilutionof the test materials.

CD4-Dependent HIV Transmission Inhibition Assay

The CD4-independent HIV transmission inhibition assay is carried outessentially as described for the CD4-independent transmission assayexcept for the use of the CD4 positive GHOST(3) X4/R5 cell line. Thiscell line is derived from the HOS (human osteosarcoma) cell line that isnegative for HIV coreceptor and CD4 expression. The cell line isengineered to express T4 (CD4), R5 and X4 via non-selectable retroviralvectors and an HIV-2 LTR hGFP construct with a hygromycin selectablemarker. The cell lines are handled and cultured as described above forthe CD4-independent HIV inhibition assay, with the exception that2.5×10⁴ GHOST(3) X4/R5 and 5×10² mitomycin C treated H9/SK-1 cells areused in the assay. Addition of compounds, mitomycin C treatment andpost-transmission washing to remove the H9/SK-1 cells are performed asdescribed above to allow comparability of the two antiviral assays.Virus replication is assessed at 24 h post infection, following 3washes, by measurement of cell-associated p24 by ELISA to ensure asingle round of infection in the presence of CD4. Compound toxicity andcell viability are assessed by XTT dye reduction. Compounds which arejudged as active in the first test are retested with or without theaddition of mucin. Testing in the presence of mucin is carried out byaddition of 200 μg/ml of porcine mucin (Sigma Chemical Co., St Louis,Mo.) in tissue culture medium to the transmission reactions.Transmission intervals and washing without replacement of mucin orcompound are carried as described above. All determinations areperformed in triplicate with serial ½ Log₁₀ dilution of the testmaterials.

Lactobacillus Assay

Lactobacillus crispatus and Lactobacillus jensenii were obtained fromthe American Type Tissue Culture Collection and grown in LactobacilliMRS broth (Difco, Fisher Scientific, Pittsburgh, Pa.). This mediumallows efficient growth of the Lactobacilli under anaerobic conditions.Bacillus stocks are produced and frozen in 15% glycerol at −80° C. foruse in the sensitivity assay. To assess the effect of compounds on L.crispatus and L. jensenii growth, 10 ml of MRS media is inoculated witha stab from the glycerol bacterial stock. The culture is placed in a GasPak CO₂ bag and incubated for 24 h at 37° C. The next day thelactobacillus cultures are diluted to an OD of 0.06 at a wavelength of670 nm. Compounds are diluted and placed into a 96 well flat bottomedplate and the Lactobacillus sp. added. Commercially availablePenicillin/Streptomycin solution at a high test concentration of 1.25U/ml and 1.25 μg/ml, respectively, are used as the positive control. Theplates are again incubated for 24 h at 37° C. in a Gas Pak CO₂ bag andbacterial growth determined by measurement of optical density at 490 nmin a molecular devices plate reader. All determinations are performedwith 6½ log dilutions from a high test concentration in triplicate.

Virus Attachment Assay

This assay is designed to detect compounds that interact with the celland block virus attachment, and/or compounds which interact with theforming attachment/fusion complex. The attachment assay is performedwith HeLa CD4 LTR β-gal cells. HeLa CD4 LTR β-gal cells are routinelycultured with the required selection antibiotics. Twenty-four h prior toinitiation of the assay the cells are trypsinized, counted and 1×10⁴cells placed in a 0.2 cm well in media without selection antibiotics. At24 h media is removed and compound in media placed on the cells andincubated for 15 min at 37° C. A known titer of the IIIB strain of HIVis then added to the wells and the incubation continued for 1 h. At theend of the incubation the wells are washed 3 times with media and theculture continued for 40 to 48 h. At termination of the assay, media isremoved and β-galactosidase enzyme expression determined bychemiluminescence per manufacturers instructions (Tropix Gal-screen™,Bedford Mass.) by a single step chemiluminescent method using a singlesolution to lyse the cells and detect β-galactosidase enzyme activity.Compound toxicity is monitored on a sister plate by XTT dye reduction.All determinations are performed in triplicate with serial ½ Log₁₀dilution of the test materials. The virus adsorption interval of 1 h issufficiently short that AZT, which requires phosphorylation to itsactive tri-phosphate form (AZT-TTP), is not active in this assay.

Fusion Assay

The fusion assay assesses the ability of compounds to block cell-to-cellfusion mediated by HIV-1 Env and CD4 expressed on separate cells. Thisassay is sensitive to inhibitors of both the gp120/CD4 interaction andinhibitors of the X4 coreceptor. First, 5×10³ HeLa CD4 LTR β-gal cellsare placed in microtiter wells and incubated overnight. The followingday the media is removed and the HeLa CD4 LTR β-gal cells are incubatedfor 1 h at 37° C. in fresh media with test compound. Following theincubation 5×10³ HL2/3 cells are added and the incubation continued for40 to 48 h. At 40 to 48 h β-galactosidase enzyme expression is detectedby chemiluminescence (Tropix Gal-screen™, Tropix, Bedford, Mass.).Compound toxicity is monitored on a sister plate using XTT dyereduction. All determinations are performed in triplicate with serial ½Log₁₀ dilution of the test materials.

P24 Antigen ELISA

ELISA kits are purchased from Coulter Electronics, and detection ofsupernate or cell-associated p24 antigen is performed according to themanufacturer's instructions. For cell-associated p24, cell lysates areproduced by lysis of the well contents in 25 to 50 μl of Coultersupplied virus lysis buffer, and assayed following 1 round offreeze/thaw. All p24 determinations are performed following serialdilution of the samples to ensure absorbances in the linear range of thestandard p24 antigen curve. The standard curve is produced usingmanufacturer supplied standards and instructions. Data are obtained byspectrophotometric analysis at 450 nm using a Molecular Devices Vmaxplate reader. Final concentrations are calculated from the opticaldensity values using the Molecular Devices Soft Max software package andexpressed in pg/ml p24 antigen.

XTT Staining for Cell Viability and Compound Cyotoxicity

TC₅₀ values for the test materials are derived by measuring thereduction of the tetrazolium dye XTT(2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide) in replicate microtiter plates containing cell and compoundwithout virus. XTT is metabolized by the mitochondrial enzyme NADPHoxidase in metabolically active cells to a soluble formazan product,allowing the rapid quantitative analysis cell viability and compoundcytotoxicity. XTT solution is prepared daily as a stock of 1 mg/mL inPBS. Phenazine methosulfate (PMS) solution is prepared at 15 mg/mL inPBS and stored in the dark at −20° C. XTT/PMS stock is preparedimmediately before use by diluting the PMS 1:100 into PBS and adding 40μl per mL of XTT solution. Fifty microliters of XTT/PMS are added toeach well of the plate and the plate incubated for 4 h at 37° C. The 4 hincubation has been empirically determined to be within the linearresponse range for MTS dye reduction with the indicated numbers of cellsfor each assay. Adhesive plate sealers are used in place of the lids,the sealed plate is inverted several times to mix the soluble formazanproduct and the plate is read at 450 nm with a Molecular Devices Vmax 96well plate format spectrophotometer.

Data Analysis

Dextran sulfate (positive) and dextran (negative) are used as controlsfor the CD4-dependent and CD4-independent HIV transmission inhibitionassays. The sulfonic acid dye Chicago Sky Blue is used as a positivecontrol for the attachment and fusion assays⁴. Commercially availablePenicillin (10,000 U/ml) Streptomycin (10.0 mg/ml) solution is used as apositive control for the Lactobacillus sensitivity assay. For eachcompound, where appropriate, an IC₅₀ (concentration inhibiting virusreplication or transmission by 50%), ID₅₀ (concentration inhibiting 50%growth of Lactobacilli) TC₅₀ (concentration resulting in a 50% reductionin cell viability) and a therapeutic index (TI: TC₅₀/IC₅₀ or ID₅₀) iscalculated by linear regression. RESULTS OF THE CD4-DEPENDENT ANDCD4-INDEPENDENT TRANSMISSION ASSAYS CD4-Independent TransmissionCD4-Dependent Assay Transmission Com- (μg/ml) Assay (μg/ml) pound Exp. #Mucin IC₅₀ TC₅₀ TI IC₅₀ TC₅₀ TI Dextran 1 − 0.316 >100 >316 Sulfate 2 −1.1 >50 >46 0.158 >50 >316 2 + 0.16 >50 >309 0.158 >50 >316 Dextran 1− >100 >100 — 2 − >100 >100 — >100 >100 — 2 + >100 >100 — >100 >100 —SPL7013 1 − 0.76 >100 >132 2 − 28.5 >100 >3.5 0.39 >100 >256 2 +1.1 >100 >90.9 0.49 >100 >205 SPL7304 1 − <0.3 >100 >316 2 −5.0 >100 >20 0.89 >10 >112 2 + 2.8 >100 >36 0.65 >100 >153 SPL7320 1 −<0.3 >100 >316 2 − 0.8 >100 >125 0.68 >100 >147 2 + 0.8 >100 >1251.15 >10  >87

Results of the Lactobacillus sp. Testing L. crispatus Compound Units(ID₅₀) L jensenii (ID₅₀) Comments Penicillin/ Dilution¹ 1:150,001:180,000 Control Streptomycin Compound SPL7013 μg/ml >100 >100 SPL7304μg/ml >100 >100 SPL7320 μg/ml >100 >100¹The starting concentrations of Penicillin and Streptomycin are 1.25U/ml and 1.25 μg/ml, respectively.

Results of the Attachment and Fusion Inhibition Assays Attachment Assay(μg/ml s) Fusion Assay (μg/ml Compound IC₅₀ TC₅₀ TI IC₅₀ TC₅₀ TI ChicagoSky Blue 0.66 >10 >15 0.79 >10 >12 0.09 >10 >111 0.62 >10 >16 SPL70130.57 >100 >175 0.71 >100 >141 SPL7304 <0.32 >100 >312 <0.32 >100 >3120.036 >1 >2778 0.56 >1 >178 SPL7320 <0.32 >100 >312 0.49 >100 >2040.049 >1 >2041

(ii) Activity of SPL7013, SPL7304 and SPl7320 Against HIV-1 RoJo and SIV89.6 pd Methods Viruses

Human immunodeficiency virus type 1 (HIV-1) strain RoJo is a low passagepediatric isolate derived in the laboratories of Southern ResearchInstitute (SRI). The SHIV89.6 pd was obtained from Mark Lewis at SRI andstocks grown in human PBMCs for antiviral testing.

PBMC Isolation and Blasting

Peripheral blood monocular cells (PBMCs) were obtained from normalhepatitis and HIV-1 negative donors by ficoll hypaque gradientseparation. Briefly, anti-coagulated blood was diluted 1:1 withDulbecco's phosphate buffered saline without Ca⁺⁺ and Mg⁺⁺ (PBS) andlayered over 14 mL of Lymphocyte separation media in a 50 ml centrifugetube. Tubes were then centrifuged for 30 minutes at 600×g. Banded PBLswere gently aspirated from the resulting interface and subsequentlywashed 2× with PBS by low speed centrifugation. The mononuclear cellswere counted, viability determined by Trypan Blue dye exclusion andresuspended in RPMI 1640 medium supplemented with 15% FBS (heatinactivated), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mLstreptomycin, and 10 μg/mL gentamycin with 2 μg/mL phytohemagluttin(PHA) at 1×10⁶ cells/mL. The cells were cultured for 48 to 72 h at 37°C., 5% CO₂. Following incubation, cells were collected bycentrifugation, washed and resuspended in RPMI 1640 supplemented with15% FBS (heat inactivated), 2 mM L-glutamine, 100 U/mL penicillin, 100μg/mL streptomycin, and 10 μg/mL gentamycin with 20 U/mL recombinantIL-2 (R & D Systems, Minneapolis, Minn.). IL-2 was included in theculture medium to maintain the cell division initiated by the PHAmitogenic stimulation. The cells were cultured in IL-2 for 72 hours andthen used for viral challenge.

PBMC Assay

Human peripheral blood mononuclear cells from a minimum of 2 donors,that have been blasted with PHA and IL-2, were counted, viabilitydetermined by Trypan Blue dye exclusion and mixed in equal ratios.Pooled donors were used to minimize the variability observed betweenindividual donors which results from quantitative and qualitativedifferences in HIV infection and overall response to the PHA and IL-2 ofprimary lymphocyte populations. The cells were resuspended at 1×10⁶cells /mL in RPMI 1640 without phenol red supplemented with 15% FetalBovine Serum (heat inactivated), 2 mM L-glutauine, 100 U/mL penicillin,100 μg/mL streptomycin, 10 μg/mL gentamycin and IL-2 (20 U/mL, R&DSystems, Minneapolis, Minn.). Fifty microliters of cells were thendistributed to the inner 60 wells of a 96 well round bottom microtiterculture plate in a standard format developed by the Infectious DiseaseResearch department of Southern Research Institute. Each plate containscell control wells (cells only), virus control wells (cells plus virus),and experimental wells (drug plus cells plus virus). Serially dilutedcompounds are added to the microtiter plate followed by the appropriatepre-titered strain of HIV- or SHIV-1. All samples were assayed intriplicate with a replicate plate without virus for the determination ofcompound toxicity. The final volume per well was 200 μL. The assay wasincubated for 6 days in a humidified atmosphere at 37° C., 5% CO₂, afterwhich supernatants were collected, for analysis of RT activity andsister plates analyzed for cell viability by MTS dye reduction. Wellswere also examined microscopically and any abnormalities noted.

MTS Staining for Cell Viability

At assay termination the assay plates were stained with the solubletetrazolium-based dye MTS (CellTiter® Reagent Promega, Madison, Wis.) todetermined cell viability and quantify compound toxicity. MTS ismetabolized by the mitochondria enzymes of metabolically active cells toa soluble formazan product, allowing the rapid quantitative analysis ofcell viability and compound cytotoxicity. This reagent is a singlestable solution that does not require preparation before use. Attermination of the PBMC assay 20 μL of MTS reagent was added per well,and the wells are incubated for 4 h at 37° C. T Adhesive plate sealerswere used in place of the lids, the sealed plate was inverted severaltimes to mix the soluble formazan product and the plate was readspectrophotometrically at 490 nm with a Molecular Devices Vmax platereader.

Reverse Transcriptase Assay

Reverse transcriptase activity was measured in cell-free supernatants.Tritiated thymidine triphosphate (NEN) (TTP) was resuspended indistilled H₂O at 5 Ci/mL. Poly rA and oligo dT were prepared as a stocksolution which was kept at −20° C. The RT reaction buffer was preparedfresh on a daily basis and consists of 125 μL 1.0 M EGTA, 125 μL dH₂O,110 μL 10% SDS, 50 μL 1.0 M Tris (pH 7.4), 50 μL 1.0 M DTT, and 40 μL1.0 M MgCl₂. These three solutions were mixed together in a ratio of 2parts TTP, 1 part poly rA:oligo dT, and 1 part reaction buffer. Tenmicroliters of this reaction mixture was placed in a round bottommicrotiter plate and 15 μL of virus containing supernatant was added andmixed. The plate was incubated at 37° C. in a water bath with a solidsupport to prevent submersion of the plate and incubated for 60 minutes.Following reaction, the reaction volume was spotted onto pieces of DE81paper, washed 5 times for 5 minutes each in a 5% sodium phosphatebuffer, 2 times for 1 minute each in distilled water, 2 times for 1minute each in 70% ethanol, and then dried. Opti-Fluor O was added toeach sample and incorporated radioactivity was quantitated utilizing aWallac 1450 Microbetaplus liquid scintillation counter.

Data Analysis

IC 50 (50%, inhibition of virus replication), TC₅₀ (50% reduction incell viability) and a therapeutic index (TI, IC₅₀/TC₅₀) are provided.AZT has been used as a relevant positive control compound for theindividual assays.

The results of the antiviral evaluations performed are summarized in thefollowing table: Summary of the Antiviral Activity of Compounds in PBMCsHIV-1 RoJo Infected SHIV 89.6pd Infected PBMCs PBMCs (μg/ml) (μg/ml)Compound IC₅₀ TC₅₀ TI IC₅₀ TC₅₀ TI SPL7013 6.1 >100 >16 0.25 >100 >400SPL7304 13.2 >100 >7.6 0.92 >100 >109 SPL7320 2.6 >100 >390.29 >100 >345 AZT (μM) 0.005 >1 >200 0.002 >1 >500

B. Activity Against Herpesviruses—HSV-1 and HSV-2 (i) Virus PlaqueReduction Assay

General Method

HSV standard strains G (HSV-2) and F (HSV-1) were used in the tests.Virus input for a 6-well plate was 100 pfu/well. HSV susceptible cellline, Vero cells, were used in the virus yield reduction assay. Forcytotoxicity test, an epithelial cell line, Hela-229 cells, wasemployed.

The antiviral effects the compounds were determined by modified plaquereduction assay. Confluent cells were washed with PBS and subsequentlyinfected with HSV (100 pfu/well) for 1 h at 37° C. and tilting every 10min. After viral inoculum was removed, infected cells were washed withPBS and overlaid with 0.5% methylcellulose in culture medium (equalvolume of 1% methylcellulose mixed with 2× culture medium). The cellswere incubated at 37° C. for 2 days for HSV-2 infection and 3 days forHSV-1. When plaque size was adequate, the cells were fixed with 10%formalin for 10 min. The plaques were subsequently stained with 0.5%crystal violet for 10 mnin. The dye was removed by washing with tapwater and left to dry in a fume hood. The plaques were then counted.

All data were generated from duplicate experiments. Mean plaque countsin test wells were compared with mean plaque counts in control wells.

EC₅₀'s (concentrations giving a 50% reduction in the plaque count of theinoculum) were calculated. Anti-viral activities and cytotoxicitymeasurements were carried out in parallel in the same strain of cells.The 50% cytotoxic concentration (CC₅₀) was defined as the compoundconcentration required to reduce the viability of mock-infected cells by50%.

Pre-Infection Treatment Method

Culture medium was removed from confluent Vero cells in 6-well plate andwashed with 1 ml of PBS. 1 ml culture medium containing the compound atconcentration of 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, and 30 μg/ml wasadded to each well and incubated at 37° C. for 1 h. Followingpre-incubation of the cells with dendrimer, the cells were then infectedwith 100 pfu/well of either HSV-1 or HSV-2. The infections wereincubated at 37° C. for 1 h with tilting every 10 min. After theinoculum was removed, the infected cells were covered with 1.5 ml of0.5% methylcellulose diluted with 2× culture medium for plaque assay.

Treatment of Infected Cells

Confluent Vero cells were washed with PBS. The cells were then infectedwith 100 pfu/well of either HSV-1 or -2 at 37° C. for 1 h. Followingremoval of viral inoculum, the infected cells were washed once with PBSand covered with 0.5% methylcellulose containing dendrimer atconcentrations of 0, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 μg/ml. Thecells were incubated at 37° C. for 2 (HSV-2) or 3 (HSV-1) days forplaque assay.

Cytotoxicity of Dendrimer

Culture medium was removed from confluent Hela-229 cells in 24-wellplates by pump. The cells were then washed once with 1 ml of PBS. 1 mlculture medium containing dendrimers at concentrations of 0, 100, 500,and 1000 μg/ml was added to each well. The cells were incubated at 37°C. incubator for 2 days. At end of incubation, the medium was removedand the cells washed with PBS. 500 μl of 0.01% neutral red (in PBS) wassubsequently added to each well, and incubated at 37° C. for 30 min. Thedye was then removed and the cells washed twice with 1 ml PBS per well.The dye was extracted by addition of 500 μl of 50% ethanol and 1%glacial acetic acid in PBS to each well and incubated at roomtemperature for 15 min with gentle shaking at 120-150 rpm. 100 μlextracted dye from each well was put into 96-well plate and theabsorbance at 550 nm was read on an ELISA reader. Table of ActivityAgainst HSV-1 and HSV-2 Compound Treatment EC₅₀ (μg/ml) CC₅₀ (μg/ml)SPL-7013 PT/HSV-2 0.61 >1000 SPL-7013 PT/HSV-1 0.93 >1000 SPL-7013INF/HSV-2 3.59 >1000 SPL-7013 INF/HSV-1 4.59 >1000 SPL-7304 PT/HSV-20.4 >1000 SPL-7304 PT/HSV-1 3 >1000 SPL-7304 INF/HSV-2 54.4 >1000SPL-7304 INF/HSV-1 50.4 >1000 SPL-7320 PT/HSV-2 0.6 >1000 SPL-7320PT/HSV-1 2.2 >1000 SPL-7320 INF/HSV-2 33.3 >1000 SPL-7320 INF/HSV-1 16.4>1000PT = Pre-infection treatmentINF = Treatment of infected cells.

(ii) Herpes Virus—2. Efficacy in an Animal Model

The efficacy of SPL-7013 was tested in a genital HSV prevention test inthe mouse (strain MS) model. A dose of 15 μl of 100 mg/ml, or 10 mg/mlof the compound was instilled into the vagina of 16 animals 20 secbefore HSV-2 infection. SPL-7013 prevented infection and disease in allanimals tested compared to controls. Total mortality in three days ofthe same number of control infected mice was used as an end-point of thetests. Table of HSV-2 genital tract infection and disease treatment inthe mouse Concentration Number of Animals Protected Against (mg/ml)Animals Disease (%) Infection (%) 100 11 11/11 (100%) 11/11 (100% 100 1212/12 (100%) 12/12 (100%  10 12 11/12 (97%) 10/12 (83%) PBS control 12 0/12 (0%)  0/12 (0%)

All animals treated 20 sec. prior to infection.

C. Human Papillomavirus (HPV). Inhibition of Human Epithelial CellUptake

The compound was evaluated for its ability to inhibit the binding anduptake of human papillomavirus virus-like particles to a humanepithelial cell line.

The compound was tested at a range of six dilutions as inhibitor ofbinding and of uptake of Papillomavirus fluorochrome-taggedVirus-Like-Particles (VLPs) of human papillomavirus type 6b. The VLPswas allowed to bind and to be taken up into the epithelial cells in thepresence of SPL-7013. Assay of binding and uptake was determined usingflow cytometry and inhibition of binding was reported as a percentagewith respect to the binding observed in the absence of inhibitor. Thetests were performed in two independent assays

Methods

Cells. The human epidermoid carcinoma cell line A431 was purchased fromthe American Type Culture Collection at passage 30 (CRL-1555, BatchF-13530) and maintained in DMEM in 10% FCS.

Human papillomavirus type 6b VLPs were grown and purified according tostandard operating procedures. Particles were labelled with afluorochrome.

The compound was re-dissolved in sterile water to the concentrations of5 mM and used fresh for the first assay before being frozen at −20° C.It was then thawed for the second assay.

Uptake Assay

A T75 flask of A431 cells was washed with 10 ml of PBS/EDTA (0.05%) for5 minutes at 37° C. before being treated with 2 ml trypsin for a further5 minutes at 37° C. DMEM/10% FCS (10 ml) was added to cells, the cellscentrifuged at 100×g for 5 minutes at RT, and re-suspended in full mediaat 3×10⁵ cells/ml in a 15 ml tube. Cell were incubated at 37° C. for 2hours with inversion every 20 minutes so as to allow re-expression ofcell surface proteins. Cells were centrifuged at 1000×g for 5 minutes atRT, and re-suspended in serum-free DMEM at 3×10⁵ cells/100 μL and 100 μLof suspension was placed in an 1.5 ml tube. Compound was added to cellsat the following six dilutions in 10 μL of PBS: 100 μM, 10 μM, 1.0 μM,100 nM, 10 nM, 1.0 nM.

Cells were incubated with compound for 30 min at 37° C. before labelledVLPs (200 ng) were added. As a positive control VLPs only, were added tocells and the negative control was cells with VLPs added but incubatedon ice. The cells were mixed and incubate at 37° C. for 2 hours. Cellswere washed once with 1 ml PBS and fixed in FACS buffer (P13S/4%paraformaldehyde). Analysis was carried out on a Coulter FACS machine.

Analysis

Results were analysed using WinLisT. Initially forward/side scatter wereused to analyse cell size and live cells were gated. The meanfluorescent intensities (MFI) of this gate was generated and used forfurther analyses. Uptake was reported as the percentage with respect tothe binding observed for VLPs alone (100%) verses cells with VLPsincubated on ice (0%).

Results

SPL-7013. Maximal inhibition of uptake (26% uptake) was observed at 1 μMwith no further inhibition at higher concentrations. This compound hasstill shown uptake inhibition at 1 μM and 10 nM. Viral uptake increasedafter this point with decreasing concentration of SPL-7013.

D. Chlamydia trachomatis Infection. Efficacy of SPL-7013 in an AnimalModel

Female mice (strain MS) were treated either in the upper or the lowergenital tract with a 15 μL instillation of 100 mg/mL solution ofSPL-7013, 20 sec prior to infection with C.trachomatis. Lower genitaltract infection is defined by isolation of the organism by culture fromvaginal swab samples collected on days 3 or 6 post challenge. For uppergenital tract infection the definition is isolation of the organism byculture from the upper genital tract tissue harvested on day 10 postchallenge. Phosphate buffer PBS treated mice were used as controls.Effects on C. trachomatis Genital Tract Infection in Mice AnimalsProtected Against Infection Group No. animals Time Lower Tract (%) UpperTract (%) Group 1. SPL-7013 16 −20 sec 8 (50%)^(a) 8 (50%) PBS 15 −20sec 1 (7%) 2 (13%) Group 2. SPL-7013 16 −20 sec 6 (38%) 8 (50%)^(a) PBS16 −20 sec 1 (7%) 1 (6%)

Persons skilled in this art will appreciate that variations andmodifications may be made to the invention as broadly described herein,other than those specifically described without departing from thespirit and scope of the invention. It is to be understood that thisinvention extends to include all such variations and modifications.

References:

-   1. Popovic, M.; Sarngadharan, M. G.; Read, E; Gallo, R. C.    Detection, isolation, and continuous production of cytopathic    retroviruses (HTLV-III) from patients with AIDS and pre-AIDS.    Science (1984), 224:497-500.-   2. Clavel, E; Guyader, M.; Guetard, D.; Salle, M.; Montagnier, L.;    Alizon, H. Molecular cloning and polymorphism of the human    immunodeficiency virus type 2. Nature (1986), 324:691-695.-   3. Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, M. R.; Schols, D.;    Herdewijn, R; Desmyter, J, De Clercq, E. Rapid and automated    tetrazolium-based colorimetric assay for the detection of anti-HIV    compounds. J. Virol. Methods (1988), 20:309-321.-   4. Clanton et al., J. AIDS. (1992), 5:771.

1. A compound of the formula I, II or III:

wherein R represents a group of the formula IV:

or a pharmaceutically acceptable salt thereof.
 2. A compound accordingto claim 1, wherein said pharmaceutically acceptable salt is selectedfrom metallic salts such as the aluminium, calcium , lithium, magnesium,potassium, sodium and zinc salts, and organic salts with organic aminessuch as N,N′-dibenzylethylenediamine, chloroprocaine, diethanolamine,ethylenediamine, dicyclohexylamine, meglumine (N-methylglucamine) andprocaine, or quaternary amines such as choline, and sulphonium andphosphonium salts.
 3. The compound SPL-7013 herein or a pharmaceuticallyacceptable salt thereof.
 4. The compound SPL-7304 herein or apharmaceutically acceptable salt thereof.
 5. The compound SPL-7320herein or a pharmaceutically acceptable salt thereof.
 6. A topicalpharmaceutical composition for prophylactic or therapeutic treatment ofsexually transmitted diseases in a human patient, which comprises acompound according to claim 1, in association with at least onepharmaceutically acceptable, topical carrier or diluent.
 7. A method forthe prophylactic or therapeutic treatment of sexually transmitteddiseases in a human patient, which comprises topical administration tothe patient of an effective amount of a compound according to claim 1.8. A method according to claim 7 wherein said disease is a vaginally orrectally sexually transmitted disease selected from HSV-1, HSV-2, HIV-1,HIV-2 and HPV infection, and Chlamydia trachomatis infection.
 9. Use ofa compound according to claim 1 in the manufacture of a medicament fortopical administration in the prophylactic or therapeutic treatment ofsexually transmitted diseases in a human patient.